The long term objective of this application is the understanding of iron and oxygen chemistries in sipunculan blood. A significant facet of this objective is characterizations of the effects of various constituents on the chemistry of hemerythrin, the non-heme iron oxygen-carrying protein in sipunculan erythrocytes. It is hoped that these factors will be easier to unravel in the more primitive sipunculans, and that their clarifications will lead to insights into these same factors in humans and to new therapeutic tools. The specific aims of this application are, (1) characterizations of the structural and functional effects of the allosteric effector, O-phosphorylethanolamine (O-PEA) on Hr, (ii) characterizations of the mechanisms of electron transfer in a naturally occurring system for reduction of methemerythrin back to its 02-binding form within the sipunculan erythrocyte, (iii) isolations and characterizations of proteins involved in iron storage and transport within sipunculan erythrocytes. Item (i) involves identifications of binding sites of O-PEA on the surface of hemerythrin and its effects on rates and mechanisms of reduction of methemerythrin. Item (ii) refers to a system consisting of transfer of electrons in the sequence, NADH leads to NADH- cytochrome b5 reductase leads to cytochrome b5 leads to methemerythrin. This system closely resembles that for reduction of methemoglobin in the human erythrocyte, which prevents methemoglobinemia. Therefore, detailed studies of the mechanism of electron transfer between cytochrome b5 and methemerythrin are proposed. A key aspect of these studies will be the test of a hypothesis regarding a recognition site for cytochrome b5 on the surface of hemerythrin. This site is proposed to be similar to that on hemoglobin. The search for iron storage and transport proteins mentioned in item (iii) will include an iron-deficient precursor of native hemerythrin and a sipunculan ferritin, analogous to that found in human reticulocytes. Practical applications which may arise from this work include new molecules for regulating redox chemistry of 02-carrying proteins and iron transport in human erythrocytes, and the use of hemerythrin as an oxygen carrier in blood substitutes.